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J Appl Physiol 95: 1799-1807, 2003. First published July 11, 2003; doi:10.1152/japplphysiol.00978.2002
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Mild renal hypertension alters run training effects on the frequency response of rat cardiomyocyte mechanics

Bradley M. Palmer, Eric A. Mokelke, Anne M. Thayer, and Russell L. Moore

Department of Kinesiology and Applied Physiology, University of Colorado at Boulder, Boulder, Colorado 80309

Submitted 23 October 2002 ; accepted in final form 11 June 2003


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 ACKNOWLEDGMENTS
 REFERENCES
 
We examined the effects of run training on the frequency dependence of cardiomyocyte mechanics and intracellular calcium concentration ([Ca2+]i) dynamics in rats with mild renal hypertension. Male Fischer 344 rats aged 2–3 mo underwent a sham operation or stenosis of the left renal artery, which increased systolic blood pressure 20–30 mmHg. Half of the rats in each group underwent treadmill run training for >16 wk. Isolated cardiomyocytes were paced at 1.0 and 0.2 Hz in 2 mM external Ca2+ concentration at 29°C. Under these conditions, negative frequency responses, i.e., decreased value with increased frequency, were recorded for peak shortening, shortening velocity, and the integral of the [Ca2+]i transient in both groups. Run training amplified the negative frequency response for the integral of the [Ca2+]i transient in both groups, but it amplified the negative frequency response for the shortening dynamics only in the normotensive sham-operated and not in the hypertensive rats. These results, as well as others for relaxation parameters, suggest that renal hypertension altered the effects of run training on the frequency response for cardiomyocyte contractile apparatus and/or passive mechanical properties, which respond to [Ca2+]i.

force-frequency relationship; relaxation-frequency relationship; calcium; treadmill

THE FORCE-FREQUENCY RELATIONSHIP of cardiac muscle refers to the change in the force of contraction induced by a change in the frequency of contraction. The force-frequency relationship in instrumented and conscious subjects has consistently been observed to be positive, i.e., increased force with increased frequency, for all species yet examined due in part to its mediation in vivo by {beta}-adrenergic stimulation (25) and by nitric oxide (15). Under laboratory conditions, however, myocardium isolated from mice and rats often (4–6, 18), although not always (2), displays a negative force-frequency relationship, whereas that from higher animals and humans normally displays a positive relationship (4–6, 25). The integrity of intracellular protein kinase signaling pathways, which include a cAMP response element modulator (14), plays a significant role in determining the capacity of the myocardium to respond to changes in frequency with modifications to contraction and relaxation (9, 25). However, other mechanisms underlying the frequency responses for contraction and relaxation also include the passive mechanical properties of the myocardium, the response of the contractile apparatus to activation by Ca2+, and intracellular Ca2+ concentration ([Ca2+]i) dynamics (12, 29).

The combined influence of Ca2+ regulatory proteins and protein kinase signaling pathways on [Ca2+]i dynamics has so far provided the primary explanation for physiological and pathological force-frequency relationships (6, 25). A positive force-frequency relationship is thought to occur when experimental conditions and Ca2+ regulatory mechanisms favor increased Ca2+ influx via L-type Ca2+ channels and increased Ca2+ uptake into and release from the sarcoplasmic reticulum (SR) as the frequency of contraction rises and Ca2+-dependent protein kinase signaling pathways activate (6, 25, 30). Evidence is mounting to suggest that a negative force-frequency relationship occurs when, as the frequency of contraction rises, experimental conditions and Ca2+ regulatory mechanisms lead to a comparatively insufficient activation of protein kinase signaling pathways (1), a diminished capacity for modification of SR Ca2+ uptake rate (4–6, 30), an increased efflux of Ca2+ from cardiomyocytes (4), and/or increased proportions of refractory L-type and SR Ca2+ channels (2, 18). In failing human left ventricle (LV), for example, the reduced density, sensitivity, and basal activity of {beta}-adrenergic receptors and receptor-associated proteins are at least partially responsible for the abnormal force-frequency relationship observed in vivo and in vitro. Refer to Alpert et al. (1) and Hasenfuss and Pieske (13) for excellent reviews of the contributions of the {beta}-adrenergic signaling pathway, SR Ca2+-ATPase (SERCA2), phospholamban, Na+/Ca2+ exchanger (NCX1) and other Ca2+-handling proteins on the altered force-frequency relationship in failing human LV.

A reduction in SR Ca2+ load with an increasing frequency of contraction has been shown to accompany the negative frequency response of rat myocardial force (4–6, 30) and cardiomyocyte shortening (4, 30) and likely underlies the negative frequency response of isolated rat LV pressure development (16, 19). Experimental pressure overload and renal hypertension in the rat further diminishes SR Ca2+ load and diastolic function largely because of reduced activity of SERCA2 (7), whose modulation by phosphorylation of PHB is then limited by the accompanying depression of {beta}-adrenergic responsiveness (22). Collectively, these characteristics of negative force-frequency relationship, diminished diastolic function, and depressed {beta}-adrenergic responsiveness provide a rationale for the use of rat myocardium after pressure overload or renal hypertension as a model for the study of similar characteristics observed in failing human myocardium.

In contrast to the effects of experimental hypertension on the force-frequency relationship and {beta}-adrenergic responsiveness, exercise training in rats has been shown to make positive the frequency response of cardiomyocyte shortening (33), improve diastolic function (34), and in some accounts increase the {beta}-adrenergic responsiveness in isolated myocardium (27, 31). It has been proposed for some time that the partial restoration of cardiac functions, which have been compromised during experimental hypertension, may be possible through physical exercise (12, 26, 27). Indeed, run training has been shown to maintain otherwise diminishing cardiac function in renal hypertensive rats (26), increase the density and/or activity of SR Ca2+ regulatory proteins that are compromised in chronic hypertension and heart failure (3, 7) and rescue {alpha}-adrenergic responsiveness in rats with mild renal hypertension (16). In addition, run training has been shown to increase the fraction of SR Ca2+ released during a single contraction in cardiomyocytes from rats with mild renal hypertension (24) and may therefore be expected to encourage a positive force-frequency response.

In this study it was hypothesized that run training in male Fischer 344 rats would encourage positive frequency responses for cardiomyocyte shortening and [Ca2+]i dynamics. We report that run training instead amplified a negative frequency response for cardiomyocyte shortening dynamics in normotensive rats but not in the renal hypertensive rats. On examining the frequency response for underlying [Ca2+]i dynamics, run training amplified the negative frequency response for the integral of the [Ca2+]i transient in both the normotensive and renal hypertensive rats. These results, as well as others pertaining to diastolic function, provide evidence that renal hypertension significantly altered the normal effects of run training on the frequency responses for the cardiomyocyte contractile apparatus and/or passive mechanical properties, which dictate shortening and relengthening dynamics in response to the [Ca2+]i transient.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 DISCLOSURES
 ACKNOWLEDGMENTS
 REFERENCES
 
Animal preparation. Male Fischer 344 rats 2–3 mo old were housed in a 12:12-h light-dark cycle, given standard rat chow and water ad libitum, and randomly assigned to a normotensive (Nt, n = 14) or hypertensive (Ht, n = 14) group. Rats in the Ht group underwent stenosis of the left renal artery using surgical hemoclips of internal diameter 0.17–0.23 cm to produce a Goldblatt (2 kidney-1 clip) model of renal hypertension as described previously (16, 24). Rats of the Nt group underwent a sham operation.

After a 4-wk recovery from surgery, systolic blood pressure was recorded in each rat by using a tail cuff sphygmomanometer (Gould Instruments, Cleveland, OH) to verify the blood pressure states of the Nt and Ht groups. Half of the rats in each group were randomly assigned to a training (Tr) or sedentary (Sed) subgroup. Rats of the Tr subgroups were treadmill trained for at least 16 wk, 5 days/wk. Running duration and intensity began at 10 min/day at 20 m/min, respectively, and were progressively increased over 8 wk to 20 min at 20 m/min plus 40 min at 26 m/min. This final duration and intensity regimen was maintained for 8–14 wk. Animal care and use were conducted under the guidelines accepted by the American Physiological Society and received prior approval from the Institutional Animal Care and Use Committee at the University of Colorado, Boulder Campus.

LV cardiomyocyte isolation. LV cardiomyocytes were obtained from the LV septal and free wall by use of methods described previously (21). All chemicals and reagents were obtained from Sigma Chemical (St. Louis, MO) except where noted. In brief, animals were heparinized (250 U ip) and then anesthetized with pentobarbital sodium (35 mg/kg ip) (Abbott Laboratories, North Chicago, IL). Hearts were rapidly excised and placed in ice-cold saline. The aorta was then cannulated, and the heart was retrogradely perfused by using a modified Langendorff perfusion apparatus, which delivered a bicarbonate-based modified Krebs-Henseleit buffer, a nominally Ca2+-free solution, and solution containing collagenase (Worthington, Freehold, NJ) and hyaluronidase. All solutions were maintained at pH 7.4 and 37°C and were bubbled with 95% O2-5% CO2 gas. The atria and right ventricular free wall were removed, leaving the LV free wall and septum, which were minced and placed in a collagenase and hyaluronidase solution. Isolated cardiomyocytes were suspended in bicarbonate-based medium 199, seeded onto laminin-coated 2-cm-diameter glass coverslips, and placed in an incubator at 37°C and 5% CO2-balance room air.

Experimental protocol. After at least 2 h incubation, coverslips were removed from the media and used to form the bottom plate of a custom flow-through chamber. The chamber was placed on the stage of an inverted microscope (Nikon Diaphot) fitted with a x40 oil immersion objective. Superfusion of a Tyrode solution (in mM: 140 NaCl, 6 KCl, 1 MgCl2, 2 CaCl2, 10 glucose, 2 pyruvate, and 5 HEPES, pH = 7.4) was maintained at 29°C. Video images of cardiomyocytes were recorded and later examined off-line for visual length and area by using NIH Image 1.41 video frame-grabbing software, and the effective width was calculated as area/length.

Cardiomyocytes were electrically paced via field stimulation by using platinum electrodes with stimulus duration of 0.5 ms and voltage of 1.5 times their threshold of stimulation (Grass Instruments, Boston, MA). Shortening and [Ca2+]i dynamics were examined after steady-state contractile function was achieved at 1.0 Hz followed by 0.2 Hz.

Measurement of shortening dynamics. Cardiomyocytes not loaded with fura-2 were examined for shortening dynamics. The positions of cardiomyocyte edges were determined by using a video edge-detection device (Crescent Electronics, Provo, UT) and recorded at 200 Hz by using an analog-to-digital converter. The recorded cardiomyocyte shortening transients were analyzed by custom-made software to determine the following characteristics: peak shortening as a percentage of resting length, time to peak shortening, maximal shortening velocity normalized to resting length, and maximal relengthening velocity normalized to resting length.

Measurement of [Ca2+]i dynamics. Cardiomyocytes were exposed to 2 µM fura-2 AM (Molecular Probes, Eugene, OR) for 5 min in the incubator at 37°C. Fluorescence of fura-2 was monitored at 510 nm and excited with a system (IonOptix, Milton, MA) fitted with optical filters of 400 and 360 nm. This choice of excitation wavelengths provided a linear relationship between fluorescence ratio (R) and free Ca2+ concentration (23). Fluorescence intensities were recorded at 200 Hz as photon counting rates by use of a personal computer. The value for cardiomyocyte fluorescence background was determined for at least one cardiomyocyte per isolation by superfusion with Ca2+-free Tyrode + 2 µM digitonin for 4 min, which released cytosolic fura 2, and the subsequent measure of fluorescence with Ca2+-free Tyrode as superfusate. Average background and fluorescence of fura-2 residing in intracellular areas inaccessible to cytosolic Ca2+ were estimated from the data acquired from the cardiomyocytes exposed to digitonin. Background and intracellular compartmentalization of fura-2 for each cardiomyocyte were then estimated on the basis of the relative dimensions of the cardiomyocyte and were therefore incorporated into the calculation of R (23).

The recorded cardiomyocyte R transients were analyzed to determine the following characteristics: resting R (Rrest), peak minus resting R (Rdiff), the integral of the R transient above Rrest (Rint), time to peak R, and the falling exponential rate constant (kfall) determined by nonlinear least-squares fitting of a single-exponential function to the falling portion of the recorded R transient (24). It should be noted that, because of a linear relationship between R and [Ca2+], the relative values and temporal characteristics for R directly reflected those for the [Ca2+]i transient.

Analysis. All analyses were performed using SPSS version 6.1 (SPSS). All data are reported as means ± SE. To test for morphological differences between the Nt and Ht groups and between the Sed and Tr subgroups, a 2 (Nt, Ht) x 2 (Sed, Tr) ANOVA was performed on all variables describing systolic blood pressure, body mass, kidney masses, cardiomyocyte dimensions, and citrate synthase activity. To test for the effects of blood pressure state, training state, and pacing frequency on cardiomyocyte shortening and [Ca2+]i dynamics, a 2 (Nt, Ht) x 2 (Sed, Tr) x 2 (1.0 Hz, 0.2 Hz) repeated-measures ANOVA was performed on all variables. For all variables, Duncan's multirange post hoc test was applied to determine significant differences at the P < 0.05 level between the Nt+Sed, Nt+Tr, Ht+Sed, and Ht+Tr subgroups at each experimental condition. Statistical significance of ANOVA results was reported at the P < 0.05 level, although P < 0.1 was interpreted to indicate neither a significant difference nor similarity (32).


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 DISCLOSURES
 ACKNOWLEDGMENTS
 REFERENCES
 
Animal model. Characteristics of the animal model used in this study are presented in Table 1. Renal hypertension was characterized by a significantly increased systolic blood pressure of ~20–30 mmHg, increased right kidney mass (~35%), and decreased left kidney mass (~40%). These results in the Ht group collectively suggested that stenosis of the left renal artery successfully reduced blood flow to the left kidney and led to significant physiological modifications consistent with a rat model of renal hypertension described by others (16, 22, 24, 30). Run training induced an increased citrate synthase activity of the plantaris muscle (~18%), decreased body mass (~12%), and increased cardiomyocyte length (~5%). These results in the Tr subgroups suggested that both peripheral and cardiac-specific adaptations to run training were similar to those reported previously (16, 17, 21, 24).


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  		Table 1. Animal morphology characteristics, plantaris muscle CSA, and cardiomyocyte dimensions

 

The isolation of cardiomyocytes precluded our measuring ventricular masses for statistical comparison. However, it is interesting to note that we did not find cardiomyocyte width in the Ht group to be increased compared with that in the Nt group. It has been most often reported that renal hypertension causes myocardial hypertrophy characterized by wider cardiomyocytes (22, 30, 35) when systolic blood pressure has been raised >50 mmHg (8, 20). However, other two-kidney, one-clip models of renal hypertension in male Fischer 344 and male Sprague-Dawley rats, which have shown increases in systolic blood pressure of only 20–30 mmHg, do not develop cardiac hypertrophy (16, 20) or increased cardiomyocyte width or area (24). Our rat model of renal hypertension would therefore be best characterized as mild or moderate renal hypertension without cardiac hypertrophy.

Frequency response of cardiomyocyte shortening dynamics. Figure 1 depicts representative shortening transients recorded from Nt+Sed (A), Nt+Tr (B), Ht+Sed (C), and Ht+Tr (D) cardiomyocytes stimulated at 1.0 and 0.2 Hz. Cardiomyocytes of all four subgroups demonstrated a significant increase in peak shortening when pacing frequency was reduced to 0.2 Hz and therefore displayed the negative frequency response expected for rat cardiomyocytes under these experimental conditions (6, 30). The frequency response of peak shortening was not found to be dependent on the blood pressure state or on the training state of the rats, as indicated by the lack of statistical significance for the blood pressure x frequency and training x frequency interactions (Fig. 2A). There was, however, a statistically significant blood pressure x training x frequency interaction for peak shortening. This interaction is illustrated in Fig. 2A, where the peak shortening of the Nt+Tr subgroup was significantly less than that of Nt+Sed at 1.0 Hz although not at 0.2 Hz, and no similar trend was evident between the Ht+Tr and Ht+Sed. Therefore, run training amplified the negative frequency response for cardiomyocyte peak shortening in the Nt group but not in the Ht group.



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  		Fig. 1. Representative cardiomyocyte shortening transients recorded at 1.0 and 0.2 Hz. A: characteristics of cardiomyocyte shortening in the normotensive (Nt), sedentary (Sed) rats demonstrated a negative frequency response under these experimental conditions, i.e., as pacing frequency was reduced, there was an increase in peak shortening, maximal shortening velocity, and maximal relaxation velocity. B: run training (Tr) in the Nt group amplified the negative frequency responses of these characteristics. C: characteristics of cardiomyocyte shortening in the hypertensive (Ht) Sed rats demonstrated a negative frequency response. D: however, run training in the Ht group did not elicit an amplification of the negative frequency responses of the shortening characteristics examined.

 


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  		Fig. 2. Characteristics of cardiomyocyte shortening transients recorded at 1.0- and 0.2-Hz pacing frequencies. A: run training in the Nt group amplified the frequency response of peak shortening but did not change the frequency response of peak shortening in the Ht group. B: there was no effect of run training on the frequency response of time to peak shortening in either group. C: run training amplified the frequency response of maximal shortening velocity in the Nt group but not in the Ht group. D: run training also amplified the frequency response of the maximal relaxation velocity in the Nt group but not in the Ht group. The numbers of cardiomyocytes in each subgroup were as follows: 95 Nt+Sed, 80 Nt+Tr, 69 Ht+Sed, and 82 Ht+Tr. *P < 0.05 for post hoc analyses between Sed and Tr subgroups at the same pacing frequency (freq). bp, Blood pressure; tr, training state. *P < 0.05 by ANOVA. NS, not significant.

 

Time to peak shortening at both 1.0 and 0.2 Hz was significantly lowered by run training in the Nt group but not in the Ht group (Fig. 2B). It is noteworthy that run training reduced the time to peak shortening in the Nt group to a value similar to that in the Ht+Sed and Ht+Tr subgroups; therefore, renal hypertension alone induced a reduction in time to peak shortening compared with Nt and thereby may have inhibited any further effect of run training on cardiomyocyte time to peak shortening. With regard to the frequency response, time to peak shortening for all four subgroups tended to rise with the lowering of pacing frequency (Fig. 2B). Cardiomyocytes of the Nt group were significantly more sensitive to the pacing frequency than those from the Ht group, as indicated by a significant blood pressure x frequency interaction. However, there was no differential effect of run training on the frequency response of time to peak shortening in the Nt and Ht groups.

Both maximal shortening velocity and maximal relengthening velocity displayed negative frequency responses (Fig. 2, C and D). However, there was no direct dependence of these frequency responses to blood pressure or training states. Run training amplified the negative frequency response of shortening velocity and relengthening velocity in the Nt group but not in the Ht group, as indicated by the statistically significant blood pressure x training x frequency interactions for these variables. These three-way interactions can be visualized in Fig. 2, C and D, as the differences in the slopes of the lines representing the frequency responses of the four subgroups. For example, the slope of the Nt+Tr subgroup is steeper than that for the Nt+Sed and indicates an amplified negative frequency response induced by run training in the Nt group. In contrast, the slope of the Ht+Tr subgroup is not as steep as that of the Ht+Sed and indicates a diminished negative frequency response induced by run training.

The negative frequency responses observed with peak shortening, maximal shortening velocity, and maximal relengthening velocity collectively suggested that there must be a concomitant negative frequency response for either 1) the contractile apparatus and passive mechanical properties that respond to the [Ca2+]i transient and/or 2) the underlying [Ca2+]i transient. As reported below, we investigated the latter possibility.

Frequency response of cardiomyocyte [Ca2+]i dynamics. Figure 3 depicts representative R transients recorded from Nt+Sed, Nt+Tr, Ht+Sed, and Ht+Tr cardiomyocytes stimulated at 1.0 Hz and 0.2 Hz. The reduction in pacing frequency from 1.0 Hz to 0.2 Hz elicited an increase in Rdiff and in Rint for all four cardiomyocyte subgroups, as indicated by the statistically significant frequency main effects for these variables (Fig. 4, A and B). The negative frequency responses for Rdiff and Rint imply a negative frequency response for SR Ca2+ release, which is dependent on the SR Ca2+ load, the proportion and conductance of excitable SR Ca2+ release channels, the sensitivity of the SR release channels to Ca2+-induced Ca2+ release, and the trigger for Ca2+ induced Ca2+ release provided by the inward L-type Ca2+ current (6). It should be noted that a negative frequency response for SR Ca2+ load has been previously reported for cardiomyocytes of male Fischer 344 rats studied under the same laboratory conditions (30).



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  		Fig. 3. Representative cardiomyocyte intracellular Ca2+ concentration ([Ca2+]i) transients at 1.0 Hz and 0.2 Hz recorded as the ratio (R) of fura-2 fluorescence. A:in the Nt+Sed group, the magnitude of the fluorescence ratio transient increased with a decrease in pacing frequency. The rate of [Ca2+]i fall was reduced with a decrease in pacing frequency. B: fluorescence ratio transients in the Nt+Tr group were similarly affected by a change in pacing frequency. C: in the Ht+Sed group, the magnitude of the fluorescence ratio transient increased and the rate of [Ca2+]i fall was reduced with a decrease in pacing frequency. D: run training in the Ht group significantly amplified the reduction in the rate of [Ca2+]i fall with a decrease in pacing frequency.

 


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  		Fig. 4. Characteristics of cardiomyocyte [Ca2+]i transients recorded as R at 1.0- and 0.2-Hz pacing frequencies. A: peak [Ca2+]i above resting [Ca2+]i (Rdiff) increased with a reduction in pacing frequency in all subgroups. The frequency response of Rdiff was not found to be dependent on blood pressure state, training state, or combination of blood pressure and training. B: the time integral of [Ca2+]i above resting [Ca2+]i (Rint) was dramatically increased by run training in the Ht group but not in the Nt group. There was no dependence of the frequency response of time to peak R on blood pressure state, training state, or combination of blood pressure and training. C: time to peak R increased with a decrease in pacing frequency. There was no dependence of the frequency response time to peak R on blood pressure state, training state, or combination of blood pressure and training. D: the exponential rate constant for [Ca2+]i fall (kfall) was reduced by run training in the Ht group but not in the Nt group. Run training amplified this positive frequency response of kfall in the Ht group but did not significantly change the frequency response of kfall in the Nt group. The numbers of cardiomyocytes in each subgroup were as follows: 29 Nt+Sed, 29 Nt+Tr, 27 Ht+Sed, and 54 Ht+Tr. *P < 0.05 for post hoc analyses between Sed and Tr subgroups at the same pacing frequency. Significant results from repeated-measures ANOVA identified at *P < 0.05.

 

The frequency response for Rdiff was not found to be significantly dependent on blood pressure, run training, or the combination of blood pressure and run training (Fig. 4A). In contrast, there was a statistical tendency for a significant training x frequency interaction (P = 0.066) for Rint, which demonstrated at least the possibility that run training amplified the frequency response for Rint (32). Indeed, the post hoc analysis of Rint implied that the integral of the [Ca2+]i transient was more sensitive to pacing frequency in the Nt+Tr and Ht+Tr subgroups than in their respective Nt+Sed and Ht+Sed subgroups. Therefore, the negative frequency response for Rint was amplified by run training.

The integral of the [Ca2+]i transient, as indicated by Rint, would be expected to directly influence peak cardiomyocyte shortening (29). In the Nt cardiomyocytes, for example, the frequency response for Rint (Fig. 4B) mirrors that for peak cardiomyocyte shortening (Fig. 2A) in both the Nt+Sed and Nt+Tr subgroups. In the Ht cardiomyocytes, however, the frequency response for Rint mirrors that for peak shortening only in the Ht+Sed subgroup and not in Ht+Tr subgroup. This observation suggests that the frequency-dependent characteristics of the contractile apparatus and/or of the passive mechanical elements responding to [Ca2+]i in the Ht cardiomyocytes were altered by run training in a manner dissimilar to that in the Nt.

At both 1.0 and 0.2 Hz, time to peak R was significantly prolonged by run training in the Ht group but not in the Nt (Figs. 3 and 4C). Run training during renal hypertension therefore had a significant effect on those Ca2+ regulatory mechanisms collectively responsible for the rate of [Ca2+]i rise; specifically, run training may have reduced SR Ca2+ load, the proportion and conductance of excitable SR Ca2+ release channels, the sensitivity of the SR release channels to Ca2+-induced Ca2+ release, and/or the inward L-type Ca2+ current in the Ht group. With regard to frequency response, the variable time to peak R was sensitive to pacing frequency, but this sensitivity to pacing frequency was not dependent on blood pressure state, training state, or the combination of blood pressure and training (Fig. 4C).

The rate of [Ca2+]i fall, kfall, was reduced with the reduction in pacing frequency in all four subgroups (Figs. 4D) and was therefore the only variable that demonstrated a positive frequency response under these laboratory conditions. The representative [Ca2+]i transients in Fig. 3 visually attest to a reduced rate of Ca2+ uptake during pacing at 0.2 Hz compared with that at 1.0 Hz. Because kfall indicates the sum of the rates of SERCA2 and NCX1 activity, the positive frequency response for kfall was likely due in part to a frequency-dependent enhancement of SERCA2 activity via the phosphorylation of phospholamban by Ca2+-dependent protein kinase signaling pathways (6, 25). Run training significantly amplified the frequency response of kfall in the Ht group but did not appreciably change the frequency response of kfall in the Nt group as indicated by the statistically significant blood pressure x training x frequency interaction (Fig. 4D). As will be discussed in more detail below, the sharp contrast between the positive frequency response for kfall (Fig. 4D) and the negative frequency response for relengthening velocity (Fig. 2D) implies that the frequency response of mechanical relengthening is strongly influenced by the contractile apparatus and/or the passive mechanical elements that respond to [Ca2+]i dynamics.

Frequency responses of contractile apparatus and passive mechanics. The mechanical sensitivity of the cardiomyocyte contractile apparatus and the passive mechanical elements to [Ca2+]i dynamics was calculated in two ways: 1) the ratio of cardiomyocyte peak shortening to Rint to indicate sensitivity during shortening and 2) the ratio of relengthening velocity to kfall to indicate sensitivity during relengthening (29). These ratios were calculated from values that were acquired simultaneously from those cardiomyocytes loaded with fura-2.

As illustrated in Fig. 5A and indicated by the statistically significant blood pressure x training x frequency interaction, the frequency response for the mechanical sensitivity during shortening was increased by run training in the Nt group and decreased by run training in the Ht. As depicted similarly in Fig. 5B, the frequency response for the mechanical sensitivity during relengthening was also increased by run training in the Nt group and decreased in the Ht. On the basis of these results it would be expected that the contractile apparatus and/or the passive mechanical properties of cardiomyocyte are normally influenced by run training in such a way as to induce an increased negative frequency response of shortening and relengthening under these laboratory conditions independent of the influence on [Ca2+]i dynamics. Run training during mild renal hypertension, however, induced a decreased negative frequency response of the contractile apparatus and/or the passive mechanical properties of cardiomyocyte.



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  		Fig. 5. Sensitivity of cardiomyocyte mechanics in response to [Ca2+]i dynamics at 1.0- and 0.2-Hz pacing frequencies. A: the ratio of cardiomyocyte peak shortening to Rint was used to indicate the sensitivity the contractile apparatus and passive mechanical properties to [Ca2+]i during cardiomyocyte shortening. The frequency response of this sensitivity during shortening was amplified by run training in the Nt group but not in the Ht. B: the ratio of relengthening velocity (Relength. Vel.) to kfall was used to indicate sensitivity during relengthening. Again, the frequency response of this sensitivity during relengthening was amplified by run training in the Nt group but not in the Ht. These results highlight that the contractile apparatus and/or the passive mechanical properties of the isolated cardiomyocyte possess intrinsic frequency responses that are independent of the frequency response of [Ca2+]i dynamics and that these frequency responses are modified by run training during normotension but not during renal hypertension. *P < 0.05 for post hoc analyses between Sed and Tr subgroups at the same pacing frequency. Significant results from repeated-measures ANOVA identified at *P < 0.05.

 


   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
DISCLOSURES
 ACKNOWLEDGMENTS
 REFERENCES
 
It has been well established that the frequency responses for cardiac contraction and relaxation are strongly influenced by [Ca2+]i dynamics and the modification of Ca2+ regulatory proteins by protein kinase signaling pathways (1, 6, 13, 25). We found in the present study that the frequency responses for rat cardiomyocyte shortening and relengthening dynamics were differentially affected by treadmill run training and mild renal hypertension in a manner that was not mimicked by the frequency responses for [Ca2+]i dynamics. Our results therefore suggest that the cardiomyocyte contractile apparatus and/or passive mechanical properties, which dictate shortening and relengthening dynamics in response to [Ca2+]i dynamics, play considerable roles in determining mechanical frequency responses at the cardiomyocyte level. Furthermore, our results suggest that run training significantly modified the frequency response of the cardiomyocyte contractile apparatus and/or passive mechanical properties and that mild renal hypertension altered these effects of run training.

The specific Ca2+ regulatory mechanisms underlying the negative frequency response for cardiomyocyte contractile functions were not elucidated in the present study but would be expected to include SR Ca2+ load (4–6), which has been shown to possess a negative frequency response in normotensive and hypertensive rat cardiomyocytes examined under similar experimental conditions (30). A negative frequency response for SR Ca2+ load is compatible with the negative frequency responses observed in the present study for Rdiff and Rint. However, a negative frequency response for SR Ca2+ load would seemingly be incompatible with the positive frequency response for kfall, which reflects the sum of the rates of SERCA2 and NCX1. We would therefore expect a positive frequency response for Ca2+ efflux by NCX1 as suggested previously (19).

The underlying Ca2+ regulatory mechanisms, although certainly responsible for the negative frequency responses of Rdiff and Rint and for the positive frequency response of kfall, could not have been fully responsible for the negative frequency responses of cardiomyocyte shortening and relengthening dynamics observed for the various experimental groups in the present study. As shown in Fig. 2A, run training in the Nt group amplified the negative frequency response for cardiomyocyte peak shortening, which could be due to 1) the amplified negative frequency response for the integral of the [Ca2+]i transient shown in Fig. 4B but may also be due to 2) the amplified negative frequency response for Ca2+ sensitivity of the contractile apparatus as shown in Fig. 5A. Run training has been shown previously to increase the basal Ca2+ sensitivity of the contractile apparatus (10, 33). Run training in the Ht group 1) did not affect the negative frequency response for cardiomyocyte peak shortening (Fig. 2A) and yet 2) induced an amplified negative frequency response for the integral of the [Ca2+]i transient (Fig. 4B). Therefore, as illustrated in Fig. 5A, the state of mild renal hypertension must have diminished or altered the run training effect on the frequency response for the contractile apparatus and/or other passive mechanical properties, which cause the cardiomyocyte to shorten in response to [Ca2+]i.

The influence of mild renal hypertension on the effect of run training was also demonstrated in diastolic function. Run training in the Nt group amplified the negative frequency response for relengthening velocity (Fig. 2D) but did not affect the positive frequency response for kfall (Fig. 4D). In contrast, run training in the Ht group did not appreciably affect the frequency response for relengthening velocity (Fig. 2D) but amplified a positive frequency response for kfall (Fig. 4D). Therefore, as illustrated in Fig. 5B, run training in the Nt and Ht groups must have differentially affected the frequency responses for cardiomyocyte Ca2+ sensitivity, stiffness, viscosity, and/or actomyosin cross-bridge off-rate, which is dependent on the cardiac myosin heavy chain (MHC) isoform and ATP availability at the myofibrils.

Of the above possibilities, we believe we can rule out the increase in Ca2+ sensitivity and changes in MHC isoform as underlying the differential effects of run training in the Nt and Ht groups. For example, we observed a shorter time to peak shortening relative to time to peak R induced by run training in both the Nt and Ht groups, which suggests an increase in Ca2+ sensitivity induced by run training in both groups (10). In addition, cardiac MHC isoform, which affects actomyosin cross-bridge off-rate, may be expected to switch from {alpha}-MHC to {beta}-MHC in the Ht group (22). However, MHC isoform does not change with run training in the male Fischer 344 rat possessing a high density of {beta}-MHC (11, 26). Therefore, the differential effects of run training on the frequency response of cardiomyocyte mechanics are not likely due to changes in myofilament Ca2+ sensitivity or MHC isoform but are likely due to changes in stiffness (34), viscosity, and ATP availability.

There are specific attributes of the renal hypertension model used in the present study that better put our findings into context of other rat models of hypertension. As reported from previous studies, cardiomyocytes isolated from rats with severe hypertension, which has been characterized by increased systolic blood pressure of >50 mmHg, cardiac hypertrophy, and increased cardiomyocyte width (8, 20), exhibit diminished shortening (22, 35) and [Ca2+]i dynamics (22). Cardiomyocytes from our model of mild renal hypertension, which was characterized by increased systolic blood pressure of 20–30 mmHg leading to no hypertrophy (16, 20) and no increase in cardiomyocyte width (24), did not exhibit any tendencies toward diminished shortening nor [Ca2+]i dynamics under these laboratory conditions. Instead, mild renal hypertension may have subtly increased contractile function, as best illustrated by the reduced time to peak shortening (Figs. 1 and 2B) and reduced time to peak R (Figs. 3 and 4C) in the Ht+Sed compared with Nt+Sed rats. Cardiomyocytes from our model of mild renal hypertension therefore demonstrated compensated or otherwise uncompromised function and provided a useful context, within which the combined effects of run training and renal hypertension on cardiomyocyte frequency responses could be interpreted without the complications of diminished basal function.

In conclusion, our data strongly suggest that mild renal hypertension altered the modifications to cardiomyocyte properties that would otherwise be normally induced by run training. However, the present study does not clarify which modifications in cardiomyocyte mechanical properties were affected by run training and altered by mild renal hypertension. On the basis of our results, further study of the effects of run training and renal hypertension on the contractile apparatus and passive mechanical properties of the myocardium is warranted and would likely include measures of the frequency dependence of the passive mechanical properties of Ca2+-activated myofilaments.


   DISCLOSURES
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
ACKNOWLEDGMENTS
 REFERENCES
 
This work was supported in part by National Heart, Lung, and Blood Institute Grants HL-40306 and HL-44146.


   ACKNOWLEDGMENTS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 ACKNOWLEDGMENTS
REFERENCES
 
We are grateful for the expert technical assistance of Alexander Hazel, M. Charlotte Olsson, and Steven M. Snyder.


   FOOTNOTES
 

Address for reprint requests and other correspondence: R. L. Moore, Dept. of Integrative Physiology, Campus Box 354, University of Colorado, Boulder, CO 80309.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 ACKNOWLEDGMENTS
 REFERENCES
 

  1. Alpert NR, Leavitt BJ, Ittleman FP, Hasenfuss G, Pieske B, and Mulieri LA. A mechanistic analysis of the force-frequency relation in non-failing and progressively failing human myocardium. Basic Res Cardiol 93, Suppl 1: 23-32, 1998.
  2. Antoons G, Mubagwa K, Nevelsteen I, and Sipido KR. Mechanisms underlying the frequency dependence of contraction and [Ca2+]i transients in mouse ventricular myocytes. J Physiol 543: 889-898, 2002.
  3. Assayag P, Charlemagne D, de Leiris J, Boucher F, Valere PE, Lortet S, Swynghedauw B, and Besse S. Senescent heart compared with pressure overload-induced hypertrophy. Hypertension 29: 15-21, 1997.
  4. Bassani RA and Bers DM. Rate of diastolic Ca release from the sarcoplasmic reticulum of intact rabbit and rat ventricular myocytes. Biophys J 68: 2015-2022, 1995.
  5. Bers DM. SR Ca loading in cardiac muscle preparations based on rapid-cooling contractures. Am J Physiol Cell Physiol 256: C109-C120, 1989.
  6. Bers DM. Excitation-Contraction Coupling and Cardiac Contractile Force. Nowell, MA: Kluwer Academic, 2001.
  7. Buttrick PM, Kaplan M, Leinwand LA, and Scheuer J. Alterations in gene expression in the rat heart after chronic pathological and physiological loads. J Mol Cell Cardiol 26: 61-67, 1994.
  8. Capasso JM, Palackal T, Olivetti G, and Anversa P. Left ventricular failure induced by long-term hypertension in rats. Circ Res 66: 1400-1412, 1990.
  9. DeSantiago J, Maier LS, and Bers DM. Frequency-dependent acceleration of relaxation in the heart depends on CaMKII, but not phospholamban. J Mol Cell Cardiol 34: 975-984, 2002.
  10. Diffee GM, Seversen EA, and Titus MM. Exercise training increases the Ca2+ sensitivity of tension in rat cardiac myocytes. J Appl Physiol 91: 309-315, 2001.
  11. Farrar RP, Starnes JW, Cartee GD, Oh PY, and Sweeney HL. Effects of exercise on cardiac myosin isozyme composition during the aging process. J Appl Physiol 64: 880-883, 1988.
  12. Fregly MJ. Effect of an exercise program on development of hypertension in rats. J Appl Physiol 56: 381-387, 1984.
  13. Hasenfuss G and Pieske B. Calcium cycling in congestive heart failure. J Mol Cell Cardiol 34: 951-969, 2002.
  14. Isoda T, Paolocci N, Haghighi K, Wang C, Wang Y, Georgakopoulos D, Servillo G, Della Fazia MA, Kranias EG, Depaoli-Roach AA, Sassone-Corsi P, and Kass DA. Novel regulation of cardiac force-frequency relation by CREM (cAMP response element modulator). FASEB J 17: 144-151, 2003.
  15. Khan SA, Skaf MW, Harrison RW, Lee K, Minhas KM, Kumar A, Fradley M, Shoukas AA, Berkowitz DE, and Hare JM. Nitric oxide regulation of myocardial contractility and calcium cycling. Independent impact of neuronal and endothelial nitric oxide synthases. Circ Res 92: 1322-1329, 2003.
  16. Korzick DH and Moore RL. Chronic exercise enhances cardiac {alpha}1-adrenergic inotropic responsiveness in rats with mild hypertension. Am J Physiol Heart Circ Physiol 271: H2599-H2608, 1996.
  17. Laughlin MH, Schaffer ME, and Sturek M. Effect of exercise training on intracellular free Ca2+ transients in ventricular myocytes of rats. J Appl Physiol 73: 1441-1448, 1992.
  18. Maier LS, Bers DM, and Pieske B. Differences in Ca2+-handling and sarcoplasmic reticulum Ca2+-content in isolated rat and rabbit myocardium. J Mol Cell Cardiol 32: 2249-2258, 2000.
  19. Maier LS, Pieske B, and Allen DG. Influence of stimulation frequency on [Na+]i and contractile function in Langendorff-perfused rat heart. Am J Physiol Heart Circ Physiol 273: H1246-H1254, 1997.
  20. Marcus KD and Tipton CM. Exercise training and its effects with renal hypertensive rats. J Appl Physiol 59: 1410-1415, 1985.
  21. Moore RL and Korzick DH. Cellular adaptations of the myocardium to chronic exercise. Prog Cardiovasc Dis 37: 371-396, 1995.
  22. Moore RL, Yelemarty RV, Misawa H, Scaduto RC Jr, Pawlush DG, Elensky M, and Cheung JY. Altered Ca2+ dynamics in single cardiac myocytes from renal hypertensive rats. Am J Physiol Cell Physiol 260: C327-C337, 1991.
  23. Palmer BM and Moore RL. Excitation wavelengths for fura 2 provide a linear relationship between [Ca2+] and fluorescence ratio. Am J Physiol Cell Physiol 279: C1278-C1284, 2000.
  24. Palmer BM, Lynch JM, Snyder SM, and Moore RL. Renal hypertension prevents run training modification of cardiomyocyte diastolic Ca2+ regulation in male rats. J Appl Physiol 90: 2063-2069, 2001.
  25. Ross J Jr. Adrenergic regulation of the force-frequency effect. Basic Res Cardiol 93, Suppl 1: 95-101, 1998.
  26. Schaible T, Malhotra A, Ciambrone G, Buttrick P, and Scheuer J. Combined effects of hypertension and chronic running program on rat heart. J Appl Physiol 63: 322-327, 1987.
  27. Scheuer J, Malhotra A, Hirsch C, Capasso J, and Schaible TF. Physiologic cardiac hypertrophy corrects contractile protein abnormalities associated with pathologic hypertrophy in rats. J Clin Invest 70: 1300-1305, 1982.
  28. Spina RJ, Ogawa T, Coggan AR, Holloszy JO, and Ehsani AA. Exercise training improves left ventricular contractile response to {beta}-adrenergic agonist. J Appl Physiol 72: 307-311, 1992.
  29. Spurgeon HA, duBell WH, Stern MD, Sollott SJ, Ziman BD, Silverman HS, Capogrossi MC, Talo A, and Lakatta EG. Cytosolic calcium and myofilaments in single rat cardiac myocytes achieve a dynamic equilibrium during twitch relaxation. J Physiol 447: 83-102, 1992.
  30. Stauffer BL, Palmer BM, Hazel A, Cheung JY, and Moore RL. Hypertension alters rapid cooling contractures in single rat cardiocytes. Am J Physiol Cell Physiol 272: C1000-C1006, 1997.
  31. Takeda N, Dominiak P, Turck D, Rupp H, and Jacob R. The influence of endurance training on mechanical catecholamine responsiveness, beta-adrenoceptor density and myosin isoenzyme pattern of rat ventricular myocardium. Basic Res Cardiol 80: 88-99, 1985.
  32. William JL, Hathaway CA, Kloster KL, and Layne BH. Low power, type II errors, and other statistical problems in recent cardiovascular research. Am J Physiol Heart Circ Physiol 273: H487-H493, 1997.
  33. Wisloff U, Loennechen JP, Falck G, Beisvag V, Currie S, Smith G, and Ellingsen O. Increased contractility and calcium sensitivity in cardiac myocytes isolated from endurance trained rats. Cardio Res 50: 495-508, 2001.
  34. Woodiwiss AJ and Norton GR. Exercise-induced cardiac hypertrophy is associated with an increased myocardial compliance. J Appl Physiol 78: 1303-1311, 1995.
  35. Yelemarty RV, Moore RL, Yu FTS, Elensky M, Semanchick AM, and Cheung JY. Relaxation abnormalities in single cardiac myocytes from renal hypertensive rats. Am J Physiol Cell Physiol 262: C980-C990, 1992.




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